Jean-François Desaphy

Ion channel pharmacology

SummaryBecause ion channels are involved in many cellular processes, drugs acting on ion channels have long been used for the treatment of many diseases, especially those affecting electrically excitable tissues. The present review discusses the pharmacology of voltage-gated and neurotransmitter-gated ion channels involved in neurologic diseases, with emphasis on neurologic channelopathies. With the discovery of ion channelopathies, the therapeutic value of many basic drugs targeting ion channels has been confirmed. The understanding of the genotype—phenotype relationship has highlighted possible action mechanisms of other empirically used drugs. Moreover, other ion channels have been pinpointed as potential new drug targets. With regards to therapy of channelopathies, experimental investigations of the intimate drug—channel interactions have demonstrated that channel mutations can either increase or decrease affinity for the drug, modifying its potential therapeutic effect. Together with the discovery of channel gene polymorphisms that may affect drug pharmacodynamics, these findings highlight the need for pharmacogenetic research to allow identification of drugs with more specific effects on channel isoforms or mutants, to increase efficacy and reduce side effects. With a greater understanding of channel genetics, structure, and function, together with the identification of novel primary and secondary channelopathies, the number of ion channel drugs for neurologic channelopathies will increase substantially.

Adaptation of Mouse Skeletal Muscle to Long-Term Microgravity in the MDS Mission

The effect of microgravity on skeletal muscles has so far been examined in rat and mice only after short-term (5–20 day) spaceflights. The mice drawer system (MDS) program, sponsored by Italian Space Agency, for the first time aimed to investigate the consequences of long-term (91 days) exposure to microgravity in mice within the International Space Station. Muscle atrophy was present indistinctly in all fiber types of the slow-twitch soleus muscle, but was only slightly greater than that observed after 20 days of spaceflight. Myosin heavy chain analysis indicated a concomitant slow-to-fast transition of soleus. In addition, spaceflight induced translocation of sarcolemmal nitric oxide synthase-1 (NOS1) into the cytosol in soleus but not in the fast-twitch extensor digitorum longus (EDL) muscle. Most of the sarcolemmal ion channel subunits were up-regulated, more in soleus than EDL, whereas Ca2+-activated K+ channels were down-regulated, consistent with the phenotype transition. Gene expression of the atrophy-related ubiquitin-ligases was up-regulated in both spaceflown soleus and EDL muscles, whereas autophagy genes were in the control range. Muscle-specific IGF-1 and interleukin-6 were down-regulated in soleus but up-regulated in EDL. Also, various stress-related genes were up-regulated in spaceflown EDL, not in soleus. Altogether, these results suggest that EDL muscle may resist to microgravity-induced atrophy by activating compensatory and protective pathways. Our study shows the extended sensitivity of antigravity soleus muscle after prolonged exposition to microgravity, suggests possible mechanisms accounting for the resistance of EDL, and individuates some molecular targets for the development of countermeasures.

Decrease in resting calcium and calcium entry associated with slow-to-fast transition in unloaded rat soleus muscle

Using fura‐2 and the manganese quenching technique, we show here that sarcolemmal permeability to cations (SP‐Ca) of slow‐twitch muscles is greater than that of fast‐twitch ones. This appears to be related to a higher expression and/or activity of stretch‐activated channels, whereas leak channel activities are similar. During hindlimb suspension (HU), we found highly correlated decreases in SPCa and resting calcium of soleus muscle toward values of extensor digitorum longus (EDL) muscle. This was significant as soon as 3 days of suspension, contrary to soleus muscle caffeine sensitivity and responsiveness that were not modified after this HU period. After 14 days of HU, SP‐Ca, resting calcium, and caffeine response of soleus muscle became similar to that normally observed in EDL muscle. These results demonstrate that the correlated decreases in SP‐Ca and resting calcium precede most functional changes due to HU. Given the known shortening of HU soleus muscle, we proposed that this could induce a decrease of SP‐Ca and a consequent reduction of resting calcium. According to the crucial role of resting cytosolic free calcium in the maintenance and the adaptation of muscle phenotype, our results suggest that slow‐to‐fast transition of HU soleus muscle is calcium dependent.

Taurine and skeletal muscle disorders.

Taurine is abundantly present in skeletal muscle. We give evidence that this amino acid exerts both short-term and long-term actions in the control of ion channel function and calcium homeostasis in striated fibers. Short-term actions can be estimated as the ability of this amino acid to acutely modulate both ion channel gating and the function of the structures involved in calcium handling. Long-term effects can be disclosed in situations of tissue taurine depletion and are likely related to the ability of the intracellular taurine to control transducing pathways as well as homeostatic and osmotic equilibrium in the tissue. The two activities are strictly linked because the intracellular level of taurine modulates the sensitivity of skeletal muscle to the exogenous application of taurine. Myopathies in which ion channels are directly or indirectly involved, as well as inherited or acquired pathologies characterized by metabolic alterations and change in calcium homeostasis, are often correlated with change in muscle taurine concentration and consequently with an enhanced therapeutic activity of this amino acid. We discuss both in vivo and in vitro evidence that taurine, through its ability to control sarcolemmal excitability and muscle contractility, can prove beneficial effects in many muscle dysfunctions.

Is oxidative stress a cause or consequence of disuse muscle atrophy in mice? A proteomic approach in hindlimb‐unloaded mice

Two‐dimensional proteomic maps of soleus (Sol), a slow oxidative muscle, and gastrocnemius (Gas), a fast glycolytic muscle of control mice (CTRL), of mice hindlimb unloaded for 14 days (HU mice) and of HU mice treated with trolox (HU‐TRO), a selective and potent antioxidant, were compared. The proteomic analysis identified a large number of differentially expressed proteins in a pool of ∼800 proteins in both muscles. The protein pattern of Sol and Gas adapted very differently to hindlimb unloading. The most interesting adaptations related to the cellular defense systems against oxidative stress and energy metabolism. In HU Sol, the antioxidant defense systems and heat shock proteins were downregulated, and protein oxidation index and lipid peroxidation were higher compared with CTRL Sol. In contrast, in HU Gas the antioxidant defense systems were upregulated, and protein oxidation index and lipid peroxidation were normal. Notably, both Sol and Gas muscles and their muscle fibres were atrophic. Antioxidant administration prevented the impairment of the antioxidant defense systems in Sol and further enhanced them in Gas. Accordingly, it restored normal levels of protein oxidation and lipid peroxidation in Sol. However, muscle and muscle fibre atrophy was not prevented either in Sol or in Gas. A general downsizing of all energy production systems in Sol and a shift towards glycolytic metabolism in Gas were observed. Trolox administration did not prevent metabolic adaptations in either Sol or Gas. The present findings suggest that oxidative stress is not a major determinant of muscle atrophy in HU mice.

Disuse of rat muscle in vivo reduces protein kinase C activity controlling the sarcolemma chloride conductance

Muscle disuse produced by hindlimb unloading (HU) induces severe atrophy and slow‐to‐fast fibre type transition of the slow‐twitch soleus muscle (Sol). After 2 weeks HU, the resting ClC‐1 chloride conductance (gCl) of sarcolemma, which controls muscle excitability, increases in Sol toward a value typical of the fast‐twitch EDL muscle. After 3 days of HU, the gCl increases as well before initiation of fibre type transition. Since ClC‐1 channels are acutely silenced by PKC‐dependent phosphorylation, we studied the modulation of gCl by PKC and serine–threonine phosphatase in Sol during HU, using a number of pharmacological tools. We show that a fraction of ClC‐1 channels of control Sol are maintained in an inactive state by PKC basal activity, which contributes to the lower gCl in control Sol compared to EDL. After 14 days of HU, PKC/phosphatase manipulation produces effects on Sol gCl that corroborate the partial slow‐to‐fast transition. After 3 days of HU, the early increase of gCl in Sol is entirely attributable to a reduction of PKC activity and/or activation of phosphatase, maintaining ClC‐1 channels in a fully active state. Accordingly, we found that HU reduces expression of PKCα, ɛ, and θ isoenzymes in Sol and EDL muscles and reduces total PKC activity. Moreover, we show that the rheobase current is increased in Sol muscle fibres as soon as after 3 days of HU, most probably in relation to the increased gCl. In conclusion, Sol muscle disuse is characterized by a rapid reduction of PKC activity, which reduces muscle excitability and is likely to contribute to disuse‐induced muscle impairment.

Therapeutic Approaches to Ion Channel Diseases

More than 400 genes are known that encode ion channel subunits. In addition, alternative splicing and heteromeric assembly of different subunits increase tremendously the variety of ion channels. Such many channels are needed to accomplish very complex cellular functions, whereas dysfunction of ion channels are key events in many pathological processes. The recent discovery of ion channelopathies, which, in its more stringent definition, encloses monogenic disorders due to mutations in ion channel genes, has largely contributed to our understanding of the function of the various channel subtypes and of the role of ion channels in multigenic or acquired diseases. Last but not least, ion channels are the main targets of many drugs already used in the clinics. Most of these drugs were introduced in therapy based on the experience acquired quite empirically, and many were discovered afterward to target ion channels. Now, intense research is being conducted to develop new drugs acting selectively on ion channel subtypes and aimed at the understanding of the intimate drug-channel interaction. In this review, we first focus on the pharmacotherapy of ion channel diseases, which includes many drugs targeting ion channels. Then, we describe the molecular pharmacology of ion channels, including the more recent advancement in drug development. Among the newest aspect of ion channel pharmacology, we draw attention to how polymorphisms or mutations in ion channel genes may modify sensitivity to drugs, opening the way toward the development of pharmacogenetics.

Muscle loading modulates aquaporin-4 expression in skeletal muscle

The purpose of this investigation was to determine the effect of hindlimb suspension (HS) on the expression of aquaporin‐4 (AQP4) in rat soleus as well as to test the hypothesis that AQP4 can be regulated in skeletal muscle and that the expression is related to the fiber metabolism.SDS‐ PAGE experiments showed that HS determined a twofold increase in the fast MHC isoforms, concomitant with a similar decrease in the relative amount of the slow MHC isoforms after only 8 days of HS. A marked induction in AQP4 mRNA was found by semiquantitative RT‐PCR. In agreement with the increased level of AQP4 mRNA, Western blot experiments show a 90% increase in the AQP4 content. Moreover, double immunofluorescence experiments demonstrated that after 8 days of suspension, the expression of AQP4 increased twofold in parallel with a similar increase in the number of fast IIA fibers and remained at the same level after 14 and 21 days of suspension. In parallel, the number of type I fibers decreased after suspension. Finally, our analysis of the transitional fibers from type I to type IIA that were found after 4 days of HS revealed that the expression of AQP4 temporally parallels that of type IIA MHC protein. Our results demonstrate that AQP4 expression can be regulated in skeletal muscle. Furthermore, AQP4 up‐regulation is associated with slow‐ to fast‐twitch fiber conversion, and thus to the glycolitic metabolism of the fiber. Our results also suggest that AQP4 is a component of the ensemble of muscle protein involved in muscle plasticity, depending on functional demand.

Gating of myotonic Na channel mutants defines the response to mexiletine and a potent derivative

Background: Myotonia and periodic paralysis caused by sodium channel mutations show variable responses to the anti-myotonic drug mexiletine. Objective: To investigate whether variability among sodium channel mutants results from differences in drug binding affinity or in channel gating. Methods: Whole-cell sodium currents (INa) were recorded in tsA201 cells expressing human wild-type (WT) and mutant skeletal muscle sodium channels (A1156T, hyperkalemic periodic paralysis; R1448C, paramyotonia congenita; G1306E, potassium-aggravated myotonia). Results: At a holding potential (hp) of −120 mV, mexiletine produced a tonic (TB, 0.33 Hz) and a use-dependent (UDB, 10 Hz) block of peak INa with a potency following the order rank R1448C > WT ≈ A1156T > G1306E. Yet, when assayed from an hp of −180 mV, TB and UDB by mexiletine were similar for the four channels. The different midpoints of channel availability curves found for the four channels track the half-maximum inhibitory value (IC50) measured at −120 mV. Thus differences in the partitioning of channels between the closed and fast-inactivated states underlie the different IC50 measured at a given potential. The mexiletine-derivative, Me7 (α-[(2-methylphenoxy)methyl]-benzenemethanamine), behaved similarly but was ∼5 times more potent than mexiletine. Interestingly, the higher drug concentrations ameliorated the abnormally slower decay rate of myotonic INa. Conclusions: These results explain the basis of the apparent difference in block of mutant sodium channels by mexiletine and Me7, opening the way to a more rationale drug use and to design more potent drugs able to correct specifically the biophysical defect of the mutation in individual myotonic patients.

Fiber type-related changes in rat skeletal muscle calcium homeostasis during aging and restoration by growth hormone

The mechanisms by which aging induces muscle impairment are not well understood yet. We studied the impact of aging on Ca2+ homeostasis in the slow-twitch soleus and the fast-twitch extensor digitorum longus (EDL) muscles of aged rats by using the fura-2 fluorescent probe. In both muscles aging increases the resting cytosolic calcium concentration ([Ca2+]i). This effect was independent on calcium influx since a reduced resting permeability of sarcolemma to divalent cations was observed in aged muscles likely due to a reduced activity of leak channels. Importantly the effects of aging on resting [Ca2+]i, fiber diameter, mechanical threshold and sarcolemmal resting conductances were less pronounced in the soleus muscle, suggesting that muscle impairment may be less dependent on [Ca2+]i in the slow-twitch muscle. The treatment of aged rats with growth hormone restored the resting [Ca2+]i toward adult values in both muscles. Thus, an increase of resting [Ca2+]i may contribute to muscle weakness associated with aging and may be considered for developing new therapeutic strategies in the elderly.